Polarization imaging is a promising tool to visualize hidden structures below the tissue surface. Analysis of these structures, for example the collagen network, can be used to assess the possible transition from normal tissue function to diseased tissue. We have developed sophisticated techniques of data analysis, based on Pearson correlation procedure, that allow to enhance the image quality and reveal regions of high statistical similarities within the noisy images, making possible characterization of subsurface structural features of biological tissues. To realize the potential of the method we have designed a user-friendly polarization imaging system that simultaneously images cross- and co-polarized light. Preliminary experiments have demonstrated that designed polarization adapter in combination with developed data analysis algorithms can provide quantitative information on collagen structure. We have incorporated this adapter into a conventional colposcope and started a clinical protocol to study tissue structures in the cervix. One of the widely discussed tools to assess the tissue environment (e.g., inside tumors) is apply specific fluorescent dyes with fluorescence lifetime (time for an electron to return from excited state to initial state) sensitive to specific conditions, ( pH, temperature, tissue oxygen content, nutrient supply, and bioenergetic status). The lifetime of such fluorophore can vary in response to changes in its immediate environment Mapping the lifetime and location of a fluorophore in tissue at different depths can be used to monitor such parameters. Toward this goal, we have developed a time-resolved lifetime imaging system for in vivo small animal studies that maps fluorophores lifetimes. The system consists of a single source-multiple detector array that scans the surface of the tissue. Using several source-detector separations, one is able to probe different depths of the medium. We have tested a novel pH sensitive dye in the near-infrared region that potentially allows to study the tumor environment below the skin. We have demonstrated that by using simplified back projections we are able to map near surface fluorescent lifetime in vivo. Combining this with the pre-calibrated lifetime response to pH, we have shown that biologically plausible, non-invasive, quantification of pH in mouse tumors can be determined. In collaboration with Dr. Capala in the Radiation Oncology Branch of NCI we continued our project on assessment and monitoring of HER2- positive cancers in the mouse model, using quantitative optical imaging. Applying novel specific probes and designed in-house instrumentation for near infrared fluorescence imaging we have been able to characterize tumors with different levels of HER2 overexpression in the cancer cells in vivo. Comparison of our results with ex vivo golden standard i.e. ELISA assay, performed on the same tumor, shows that our methodology of data analysis, based on compartmental ligand-receptor model, allows to quantify HER2 overexpression in vivo from a series of fluorescence images of the tumor. We have got promising results from application of our method to monitor changes in the tumor due to its treatment with a known anti-cancer drug 17-DMAG in the mouse model. To take into account effects of light scattering on observed signal from abnormalities, deeply embedded in tissues, one needs theoretical model of photon migration. In particular, to extract intrinsic fluorescence lifetime of a fluorophore such specific analytical model, based on the random walk theory, has been developed in collaboration with CIT (Drs. Weiss and Pajevic). Application of this model in the reconstruction algorithm allowed us to obtain accurate estimates of the lifetimes and depths of the fluorophores inside tissue like phantoms. Another optical imaging modality of interest to the group is Two-Photon Microscopy. Colleagues at NHLBI have recently developed a new system for Two-Photon imaging based on the Total Emission Detection (TED) principle. Here instead of using a standard TED system where only transmitted or reflected light is collected the TED Two Photon system captures all light emitted from the sample. Researchers in this group were contacted to model Two Photon emission and evaluate the systems performance. Using our Monte- Carlo simulation code we were able to evaluate different aspects of Two-Photon Imaging and the related field of Fluorescence Correlation Spectroscopy in collaboration with Section on Cell Biophysics at NICHD. The oncology community is testing a number of novel targeted approaches for use against a variety of cancers. With regard to monitoring vasculature, it is desirable to develop and assess noninvasive and quantitative techniques that can not only monitor structural changes, but can also assess the functional characteristics or the metabolic status of the tumor. We are testing three potential noninvasive imaging techniques to monitor patients undergoing an experimental therapy: infrared thermal imaging (thermography), laser Doppler imaging (LDI) and multi-spectral imaging. These imaging techniques are being tested on subjects with Kaposi s sarcoma (KS), a highly vascular tumor that occurs frequently among people infected with acquired immunodeficiency syndrome (AIDS). Cutaneous KS lesions are easily accessible for noninvasive techniques that involve imaging of tumor vasculature, and they thus represent a tumor model in which to assess certain parameters of angiogenesis. The KS studies are ongoing clinical trials under four different NCI protocols. We have shown that our multi-modality techniques can non-invasively monitor the functional properties of the tumor and surrounding tissues and has the potential to predict treatment outcomes. We have found that quantitative results can be obtained if the underlying skin structures are being taken into account. In order to obtain those structures, we have developed a spectral domain Optical Coherence Tomography (OCT) system, which gives 3D images of the skin. We also developed a novel data analysis tool, which is based on Principal Component Analysis, which allows us to obtain blood volume and oxygenation values in real time. In combination with OCT, we have recently shown quantitative results of blood volume and oxygenation, which were obtained in real time. High-resolution confocal laser microscopy is an intensively active field in modern bioimaging technologies because this technique provides sharp, high-magnification, three dimensional imaging with submicron resolution by non-invasive optical sectioning and rejection of out-of-focus information. We have developed a simple fiber-optic confocal microscope with nanoscale depth resolution beyond the diffraction barrier. We are extensively working to increase the acquisition time for data collection in 3D.